Abstract
Facioscapulohumeral muscular dystrophy (FSHD) is among the most prevalent of the adult-onset muscular dystrophies. FSHD causes a loss of muscle mass and function, resulting in severe debilitation and reduction in quality of life. Currently, only the symptoms of FSHD can be treated, and such treatments have minimal benefit. The available options are not curative, and none of the treatments address the underlying cause of FSHD. The genetic, epigenetic, and molecular mechanisms triggering FSHD are now quite well-understood, and it has been shown that expression of the transcriptional regulator double homeobox 4 (DUX4) is necessary for disease onset and is largely thought to be the causative factor in FSHD. Therefore, we sought to identify compounds suppressing DUX4 expression in a phenotypic screen using FSHD patient-derived muscle cells, a zinc finger and SCAN domain-containing 4 (ZSCAN4)-based reporter gene assay for measuring DUX4 activity, and ∼3,000 small molecules. This effort identified molecules that reduce DUX4 gene expression and hence DUX4 activity. Among those, β2-adrenergic receptor agonists and phosphodiesterase inhibitors, both leading to increased cellular cAMP, effectively decreased DUX4 expression by >75% in cells from individuals with FSHD. Of note, we found that cAMP production reduces DUX4 expression through a protein kinase A-dependent mode of action in FSHD patient myotubes. These findings increase our understanding of how DUX4 expression is regulated in FSHD and point to potential areas of therapeutic intervention.
Highlights
Facioscapulohumeral muscular dystrophy (FSHD) is among the most prevalent of the adult-onset muscular dystrophies
To ensure suitability of these FSHD type 1 myoblasts, they were sent to the University of Iowa Diagnostic Laboratories and were confirmed to have short 4q35 EcoRI fragments (21 and 27 kb, respectively) on a 4qA background by Southern blotting, indicating that these myoblasts are from FSHD type 1 patients
We examined mRNA expression levels of the previously identified Dux4-dependent genes, zinc finger and SCAN domain– containing 4 (ZSCAN4), TRIM43, MBD3L2, and LEUTX (22). mRNA levels for all four genes were below detection limits (Ct Ͼ 35) in Skmdc myoblasts and myotubes until day 7 post-differentiation when using standard Quantitative PCR (qPCR) methods
Summary
We established our assays with two previously uncharacterized myoblast isolates from FSHD type 1 patients obtained from the Coriell Institute, namely GM17940 (FSHD940) and GM17869 (FSHD869). Immunostaining for Dux was performed, and Dux4-positive nuclei exhibiting the telltale sentinel nuclei and gradient Dux expression pattern (21) were observed as early as day 5 post-differentiation in FSHD940 myotubes and by day 7 in the FSHD869 myotubes under the optimized conditions (Fig. 1D). Given the variegated expression pattern and the expected low percentage of nuclei positive for Dux by immunostaining in the FSHD cultures, we sought to identify more sensitive measures of Dux activity. To this end, we examined mRNA expression levels of the previously identified Dux4-dependent genes, ZSCAN4, TRIM43, MBD3L2, and LEUTX (22). Expression of TRIM43, MBD3L2, and LEUTX mRNA was induced to a greater extent in FSHD940 and FSHD869 myotubes compared with Skmdc myotubes at day 7 (Fig. 1E and Fig. S1B)
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